Microfluidic metering and delivery system

ABSTRACT

This invention provides devices, systems, and methods for performing point-of-care, analysis, including multiplexed analysis, of a biological fluid analyte, such as blood. The invention includes a cartridge for collecting the biological fluid analyte. The cartridge is configured to be inserted into an assay reader, in which one or more assay reactions may be performed. The assay reader is designed to read and report the results of the one or more assay reactions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/151,062, filed Oct. 3, 2018, which claims the benefit of priority toU.S. Provisional Application No. 62/571,178, filed Oct. 11, 2017, bothof which are hereby incorporated by reference in their entirety.

BACKGROUND

Point-of-care (POC) testing refers to performing medical diagnostictests at the time and place (“point of care”) that the patient is beingtreated. Point-of-care testing is advantageous over traditionaldiagnostic testing where patient samples are sent out to a laboratoryfor further analysis, because the results of traditional diagnostictests may not be available for hours, if not days or weeks, making itdifficult for a caregiver to assess the proper course of treatment inthe interim.

Although some POC testing devices are available, they typically sufferfrom one or more serious drawbacks. For example, many POC testingdevices can analyze only one target analyte at a time. And while somePOC testing devices can perform multiplexed analysis, e.g., by testingmultiple analyte targets in one test cartridge, such POC devicestypically suffer from serious drawbacks, such as the inability toprecisely control the volume of blood sample dispensed for eachanalysis, which adversely affects the accuracy of the POC testing.

Designing POC testing devices for in-home is particularly challenging,because such devices are often operated by people with limited trainingor no training at all. Current systems can often require the user tofollow multiple steps of operations of multiple separated parts(pipettes, test strips, etc.). User-introduced errors can easily causeinaccurate or failed assays. To avoid user-introduced errors, currentPOC devices separate sample collection, sample preparation, and theassay to avoid common problems such as timing inconsistencies,inaccurate sample volume, air bubble formation, and other issues.However, this approach presents other challenges. For example, samplecollection is often done using plastic pipettes or glass capillariesthat need to be filled up to a given mark. Accuracy of an assay dependson the accuracy of a sample volume, which in turn relies on relies on auser's ability to consistently and accurately fill a capillary orpipette to collect the appropriate sample volume. One of the biggestproblems relating to accurate sample collection is the creation ofundesirable air bubbles during the collection process. It can bedifficult to fill the capillary in a single motion, and thus users oftenuse several stop-and-fill motions, which may temporarily leave the tipof the capillary exposed to air rather than the desired fluid analyte(e.g., blood). When this happens, air bubbles can then get into thecapillary or pipette and prevent the collection of the appropriatevolume of fluid analyte, thereby introducing errors in the samplevolume.

In addition to problems associated with inaccurate sample volumecollection, additional problems may arise when conducting the assay orassays associated with a conventional POC testing system. For example,after a sample is collected, POC systems can require the user tomanually dispense the blood sample from the pipet or capillary to thecartridge that can perform the assay. In this step, additionaluser-introduced error such as mis-aiming, touching the assay pad, orincomplete or prolonged dispensing can further adversely affect theaccuracy assay results. For instance, in most point-of-care (POC)testing systems for blood samples, certain sample preparation steps needto be performed prior to a final chemical reaction that provides thetest result. These sample preparation steps may include complexpreparation steps such as plasma separation, cell lysis, or others,depending on the assay. The time required to complete such complexpreparation steps may be comparable to the time required for blood toundergo undesirable clotting, which further introduces error into theassay results. Thus, it would be desirable to have a POC system thatprecisely controls the amount of analyte that is collected, minimizesthe analyte collection time to avoid undesirable side reactions (e.g.,blood clotting); and controls the dispensing time during which thetarget analyte undergoes chemical reactions with the assay chemicals toprovide an assay result.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present disclosure will now be described hereinafter,by way of example only, with reference to the accompanying drawings inwhich:

FIG. 1 provides a schematic drawing of a system comprising a cartridgeand a reader according to one embodiment of the invention;

FIG. 2A-2C illustrates an embodiment of the cartridge of themicrofluidic device;

FIG. 3A-3B illustrate an embodiment of a physical compression of themetering stack and assay stack when the cartridge is inserted into anassay reader;

FIG. 3C-3D illustrate an embodiment using moving pins to compress themetering stack and assay stack when the cartridge is inserted into anassay reader;

FIG. 4A-5B illustrate embodiments of channels within a metering stack;

FIG. 6 illustrates an embodiment of a layer of the metering stack thatincludes a mesh or porous material within a portion of the layer;

FIG. 7A-7C illustrate embodiments of channels with various positions ofventing holes;

FIG. 8 illustrates various layers of a metering stack, according to someembodiments;

FIG. 9 illustrates various layers of an assay stack, according to someembodiments;

FIG. 10 illustrates a cartridge and an assay reader, according to someembodiments of the invention;

FIG. 11 illustrates a cartridge inserted into an assay reader accordingto an embodiment of the invention;

FIG. 12 illustrates a metering stack of a cartridge according to oneembodiment of the invention;

FIGS. 13A-B illustrates an assembled metering stack according to oneembodiment of the invention;

FIG. 14A illustrates a metering stack and an assay stack according toone embodiment of the invention; FIG. 14B shows a cross-sectional viewof a metering stack and assay stack according to one embodiment of theinvention;

FIG. 15A-D provides a schematic illustration of the operation of acartridge according to some embodiments of the invention;

FIG. 16A shows a longitudinal cross sectional view of an assay readeraccording to one embodiment of the invention; FIG. 16B shows alongitudinal cross sectional view of an assay reader with an insertedcartridge according to one embodiment of the invention;

FIG. 17A shows a transverse cross-sectional view of a reader accordingto one embodiment of the invention; FIG. 17B shows a transverse crosssectional view of an assay reader with an inserted cartridge accordingto one embodiment of the invention.

FIG. 18 shows a block diagram of the sensor system of assay reader,according to an exemplary implementation of the invention.

FIG. 19 shows a flow chart illustrating a method of using the assaysystem according to an exemplary implementation of the invention.

FIG. 20 shows a flow chart illustrating a method of manufacturing acartridge according to one exemplary implementation of the invention.

DETAILED DESCRIPTION

This invention relates to a microfluidic device for rapid point-of-carecollection of a fluid analyte and multiplex analysis thereof. Thisinvention also provides methods and systems for using a microfluidicdevice to provide rapid multiplexed analysis of a fluid analyte. Thepresent disclosure generally provides methods for a microfluidic devicefor sample collection and testing. Some of the devices described hereincomprise a sample collection cartridge. The sample collection cartridgecan be used in combination with an assay reader for point-of-care (POC)diagnostics as described herein.

One aspect of this invention is to provide a cartridge for collecting atarget analyte for testing. The cartridge includes a metering stackconfigured to receive and distribute a target analyte along a firstchannel. The first channel has a bottom that includes a porous or meshmaterial and one or more venting holes located along the first channel.The cartridge further includes an assay stack comprising at least oneassay component that includes one or more assay pad. Each assay padcomprises a reagent for performing an assay on the target analyte or aportion thereof. The cartridge also includes a spacer layer disposedbetween the metering stack and the assay stack. The spacer layerprovides a gap between the metering stack and the assay stack thatprevents the target analyte from flowing from the metering stack intothe assay stack when the cartridge is in an uncompressed state. Theporous or mesh material permits the target analyte to flow from themetering stack to the assay stack only when the cartridge issufficiently compressed to bring the metering stack in contact with theassay stack.

Another aspect of this invention is to provide an assay reader. Theassay reader includes a chamber configured to receive a cartridge. Theassay reader also features a compression mechanism configured tocompress the cartridge when or after the cartridge is inserted into thechamber after collection of a target analyte. This causes one or moreassay reactions to start within the cartridge. The assay reader alsoincludes a detection system for measuring a signal change correspondingto the one or more assay reactions.

The invention also provides a system for multiplexed analysis of atarget analyte. The system comprises a cartridge which includes ametering stack configured to receive and distribute the target analytealong a first channel. The first channel has a bottom that comprises aporous or mesh material. The system also includes an assay stackcomprising at least one assay component. Each assay component includesone or more assay pads that contain a reagent for performing an assay onthe target analyte or a portion thereof. The cartridge in the systemfurther includes a spacer layer disposed between the metering stack andthe assay stack. The spacer layer provides a gap between the meteringstack and the assay stack that prevents the target analyte from flowingfrom the metering stack into the assay stack when the cartridge is in anuncompressed state. In addition, the porous or mesh material permits thetarget analyte to flow from the metering stack to the assay stack onlywhen the cartridge is sufficiently compressed to bring the meteringstack in contact with the assay stack. The system further includes anassay reader that includes a chamber configured to receive the cartridgeand a compression mechanism configured to compress the cartridge when orafter the cartridge is inserted into the chamber after collection of thetarget analyte. This causes one or more assay reactions to start at thesame time within the cartridge. The system further includes a detectionsystem for detecting a signal change corresponding to the one or moreassay reactions.

In yet another aspect, the invention provides a method of performing aplurality of assays. The method includes receiving a target analyte intoa first channel in a cartridge, and inserting the cartridge into anassay reader, thereby compressing the cartridge to expose at least onecomponent of the target analyte stored in a first channel to one or moreassay pads. This causes the assay reactions to start. The method alsoincludes the step of detecting one or more signal changes associatedwith the one or more assay reactions.

The invention also provides a method of fabricating a cartridge. Themethod comprises obtaining a first layer comprising (1) a layer ofpolymeric material with a channel formed therein, wherein the channelcomprises at least one venting hole disposed along the channel, (2) aporous or mesh material attached on a bottom surface of the polymericmaterial such that the channel is bounded on a bottom surface by theporous or mesh material. The method also comprises the steps ofobtaining a second layer comprising one or more assay pads, eachcomprising a porous material capable of absorbing analyte from thebottom of the channel, and a reagent for performing an assay on thetarget analyte or a portion thereof. The method also includes the stepsof obtaining a compressible intermediate layer and combining the firstlayer, compressible intermediate layer, and second layer in a cartridgehousing such that the compressible intermediate layer separates thefirst layer and the second layer when the compressible intermediatelayer is in an uncompressed state, and the channel is aligned with theone or more assay pads in a direction perpendicular to the first layer.

The invention also provides a method of fabricating a cartridge thatcomprises combining a first layer, a compressible intermediate layer,and a second layer in a cartridge housing. The first layer comprises (1)a layer of polymeric material with a channel formed therein and (2) aporous or mesh material attached on a bottom surface of the polymericmaterial such that the channel is bounded on a bottom surface by theporous or mesh material. The second layer comprises one or more assaypads, each comprising a reagent for performing an assay on the targetanalyte or a portion thereof. The compressible intermediate layercomprises a compressible material that separates the first layer and thesecond layer when the compressible intermediate layer is in anuncompressed state. When the first layer, compressible intermediatelayer, and second layer are stacked in the cartridge housing, thechannel is aligned with the one or more assay pads in a directionperpendicular to the first layer.

Any of the features, components, or details of any of the arrangementsor embodiments disclosed in this application, including withoutlimitation any of the cartridge embodiments and any of the testing orassay embodiments disclosed below, are interchangeably combinable withany other features, components, or details of any of the arrangements orembodiments disclosed herein to form new arrangements and embodiments.

In one aspect, this invention provides an easy-to-use and fullyintegrated POC testing device that allowed for multiplexed analysis of atarget analyte. As described herein, the POC testing device simplifiestarget analyte sample collection and obviates problems associated withconventional POC testing devices, including unwanted bubble formation,inaccuracy in collected sample volumes, and uncertainty in targetanalyte dispensing time when the assay is conducted. In addition, thePOC testing devices according to the invention contain few moving partsand do not employ mechanical devices, such as plungers or valves, forcollecting the target analyte samples. In this way, the POC testingdevices of the invention are more mechanically robust and minimize thechance for user errors.

FIG. 1 shows a POC testing device according to one exemplary embodimentof the invention. The POC testing device comprises a cartridge 100 andan assay reader 110. As described herein, cartridge 100 is used tocollect the target analyte. The collection process also distributes thetarget analyte within cartridge 100. After the target analyte iscollected in cartridge 100, the user inserts cartridge 100 into assayreader 110. As described herein, the act of inserting cartridge 100 intoassay reader 110 results in the compression of cartridge 100, therebycausing at least one component of the target analyte to be distributedto a plurality of assay pads. In this way, the act of insertingcartridge 100 into assay reader 100 commences a plurality of assayreactions that provide information regarding the contents of the targetanalyte. As described herein, assay reader 110 is equipped with adetection system that is used to detect the results of the assayreactions that occur at the assay pads of cartridge 100. The detectionsystem is not particularly limited and may be a detection system whichcauses a measurable signal change as the result of an assay reaction.Non-limiting examples of suitable detection systems include optical andelectrochemical detection systems as described herein.

FIG. 2A illustrates a top view of an embodiment of a cartridge 200. InFIG. 2A, cartridge 200 includes a housing 201 attached to a handle 202.In general, cartridge 200 is designed to be easy to handle by the userand to provide a protective shell for the microfluidic distributionsystem and assay components housed within cartridge 200. In general,suitable materials for housing 201 and handle 202 include polyolefiniccompounds, such as polyethylene, polypropylene, and other polymericresins or compounds that are amenable to sterilization procedures knownin the medical device manufacturing art, e.g., exposure to ethyleneoxide gas. During sample collection, cartridge 200 is brought intocontact with a target analyte (e.g., blood). The target analyte is drawninto channel 203 and via channel opening 204 by capillary action. Insome embodiments, channel 203 comprises a plurality of receivingchambers located along channel 203. In preferred embodiments, eachreceiving chamber is positioned between two venting holes, whichfacilitate the division of the target analyte in the channel intomultiple aliquots which flow to the assay pads in the assay stack. Itshould be recognized that the channel opening 204 can function as aventing hole and that neighboring receiving chambers can share a commonventing hole between them. The venting holes, in combination with theporous or mesh material described herein, prevent unwanted bubbleformation as the target analyte is drawn into the receiving chambers.FIG. 2B illustrates a bottom view of an embodiment of the cartridge 200.In FIG. 2B, the bottom portion of housing 201 comprises a plurality ofassay detection ports 206 aligned with channel opening 204. The assaydetection ports 206 permit the assay results to be interrogated, forexample, by optical detection methods as described herein. In addition,the bottom portion of housing 201 may comprise plurality of holes 207,which are additional assay detection ports that may be used with assaycomponents and microfluidic channels that are arranged in acorresponding configuration.

FIG. 2C provides an exploded view of the components of the cartridge200, according to one embodiment of the invention. In FIG. 2C, the outershell of cartridge 200 comprises a handle 202, bottom housing portion227, and a cap 223 that is equipped with a slot 228. The bottom housingportion 227 can be a cuboid shape enclosure with one open side. Theenclosure shape of the bottom housing portion 227 protects thecomponents within the interior chamber and can avoid accidentalactuation of the device. The cap 223 can fit to the open side of thebottom housing portion 227 and have a shape and size that corresponds tothe open side of the bottom housing portion 227. When the bottom housingportion 227 and cap 223 of the housing are assembled together, aninterior chamber can be formed for enclosing other components of thecartridge within the interior chamber. In other embodiments, the cap 223and bottom housing portion 227 do not form an enclosure with an interiorchamber and can be rigid structures positioned on the top of a meteringstack and bottom of an assay stack, which are described herein.

In preferred embodiments, bottom housing portion 227 and cap 223 can beformed of a material to provide a rigid structure to the cartridge 200.For example, the bottom housing portion 227 and the cap 223 can be aplastic material, as described herein. The bottom housing portion 227and cap 223 can be moveable or non-moveable with relation to each other.In preferred embodiments, when cartridge 200 is inserted into an assayreader, the components within the interior chamber are compressed tocause at least one portion of the collected target analyte to bedelivered to a plurality of assay components.

In some embodiments, the cartridge does not comprise a cap and bottomhousing portion. In such embodiments, the cartridge does not include thehousing 201 (see FIG. 2A) and the metering stack and assay stack can beinserted into an assay reader without an enclosure around it.

As shown in FIG. 2C, cartridge 200 can include a metering stack 224, aspacer material 225, and an assay stack 226. The metering stack 224 canbe used to collect a sample of the target analyte (e.g., blood) and theassay stack 226 comprises assay components necessary for the test to becarried out as discussed in detail herein. As used herein, the term“metering” refers to collecting a liquid sample of a target analyte anddelivering one or more predetermined volumes of at least a portion ofthe target analyte to the assay components for further analysis via theassay components contained in the assay stack. When assembled into acartridge, the metering stack 224, a spacer material 225, and an assaystack 226 can arranged in a stack.

The spacer material 225 is a compressible layer that may be positionedbetween the metering stack 224 and assay stack 226 as shown in FIG. 2C.In an embodiment, the spacer layer 225 may be a flexible material thatcan be compressed in the vertical direction when the cartridge isinserted into the assay reader and the metering stack 224 is moved intocontact with or close proximity to the assay stack 226. In someembodiments, the spacer layer 225 can be a flexible material, such asfoam, rubber, porous polymer, metal, cotton, or other bending, folding,or moving mechanisms such as a clamp or spring. In some embodiments, themetering and assay stacks are initially separated by an air gapmaintained by the spacer layer 225. In certain embodiments, spacermaterial 225 is physically affixed to another layer, such as meteringstack 224 or assay stack 226 before the layers of the cartridge arebrought together. Typically, the metering and assay stacks remainseparated throughout the sample collection process. In such embodiments,the separation between the metering stack and the assay stack prevent achemical reaction from starting during the target analyte collectionstep. When the spacer material 225 is compressed, the metering stack 224and assay stack 226 can come into contact with or brought into closeproximity to each other.

In preferred embodiments, when the metering stack is fully filled withthe target analyte, the cartridge is inserted into an assay reader.Preferably, the material that is used for the top surface of channel 230is sufficiently transparent so that a user can determine by visualinspection when the channel 230 is filled and the cartridge is ready forinsertion into the assay reader. The assay reader is configured toaccept the assay reader and comprises a mechanism that compresses thespacer layer, thereby pushing the metering stack and assay stacktogether when the cartridge is inserted into the assay reader. Thecompression of the spacer layer causes a predetermined volume of atleast a portion of the collected target analyte to flow to assaycomponents in the assay stack. In this way, the act of compressing themetering and assay stacks together can, in certain embodiments, providea well-defined point in time that marks the start of the assays of theassay components in the assay stack.

In some embodiments, the metering stack and assay stack can be pushedtogether inside the assay reader by non-movable physical constraints asillustrated in FIGS. 3A-B. FIGS. 3A-B illustrate an embodiment in whichphysical compression of the metering stack 304 and assay stack 306occurs when cartridge 300 is inserted into an assay reader 307. FIG. 3Aillustrates the cartridge 300 and assay reader 307 before the cartridge300 is inserted into the assay reader 307 in the direction indicated thearrow in FIG. 3A. FIG. 3B illustrates the cartridge 300 after it hasbeen inserted into a slot of the assay reader 307. The positioning of atop portion 301 a of the assay reader slot with respect to a bottomportion 301 b of the slot causes the metering stack 304 to movevertically within the cartridge 300, thereby compressing the spacermaterial 305. If desired, the top portion 301 a may be sized to compressonly the portion of the metering stack that is directly above the assaycomponents in the assay stack. This may be achieved, for example, bysizing the top portion 301 a such that it slides into a slot in the cap303 of the cartridge (e.g., slot 228 in FIG. 2C). If desired, the assayreader slot may be shaped to limit the degree to which cartridge 300 maybe inserted into the assay reader 307. For example, once bottom housingportion 302 is fully inserted, it may interact with lip 308 to preventfurther insertion of cartridge 300 into assay reader 307. Once thespacer material 305 is compressed from the insertion of cartridge 300into assay reader 307, the collected target analyte flows from themetering stack 304 to the assay components in the assay stack 306.

Alternatively, the metering stack 304 and assay stack 306 can be pushedtogether by one or more moving parts in the assay reader, non-limitingexamples of which include pushing pins, or movable blocks actuated by amotor, servo, air pressure, magnetic force, electro-magnetic force,manual force exerted by the user, or other moving mechanisms.Combinations of these mechanisms are also expressly contemplated by theinvention. In some embodiments, the metering stack 304 and assay stack306 can be pushed together by one or more moving parts in the assayreader such as a plunger pressor using electromechanical forces in theassay reader.

FIGS. 3C-3D illustrate an embodiment of moving pins used to compress themetering stack and assay stack when the cartridge is inserted into anassay reader. As illustrated in FIGS. 3C-3D, moving pins can be used tocompress the metering stack 354 onto the assay stack 356. FIG. 3Cillustrates the cartridge with the base 352 and cap 353 within the assayreader 357. Moving pins 372 can be used within the assay reader 357 tocompress the metering stack 354 and spacer material 355 so that themetering stack 354 can come into contact with the assay stack 356. Themoving pins 357 can move downward in the direction of arrow shown inFIG. 3C to compress the metering stack 354 onto the assay stack 356. Insome embodiments, the moving pins can pass through a portion of the cap353 as shown in FIG. 3D. As illustrated in FIGS. 3C-3D, in someembodiments, the moving pins can move vertically within the assay reader357.

In some embodiments, the target analyte is blood, and the cartridge canbe used to collect a sample of blood from a skin prick and deliver thesample to the assay stack consistently with minimal user intervention.The user, with a regular pricking lancet, can elicit bleeding in asuitable body site such as a fingertip, palm, hand, forearm, belly etc.Once a drop of blood of sufficient volume is on the skin, the user cancollect it by touching the tip of the cartridge to the blood drop. Oncethe metering stack is fully filled with blood, the user can insert thecartridge into the assay reader, which triggers the delivery of theblood sample to the assay stack. In some embodiments, this can beperformed by a patient, administrator, or healthcare provider. The bloodcollection and testing as described herein does not have to be performedby a trained heath care professional.

In addition, the cartridge design can allow for dispensing differentpre-defined volumes of blood sample to multiple assay locations, withoutusing any moving parts such as pumps or valves in the cartridge or inthe assay reader. This increases the accuracy and flexibility of amultiplexed quantitative POC analysis, while reducing the complexity andcost of the cartridge and the assay reader.

Typically, as illustrated in FIG. 2C, the metering stack 224 comprises achannel 230 to contain the target analyte (e.g., blood sample). Incertain embodiments, the channel can hold a volume of target analyte inthe range of about 0.5 to about 100 μl, about 5 μl to about 90 μl, about10 to about 80 μl, about 20 μl to about 60 μl or about 30 μl to about 50μl. The volume of the target analyte can be controlled by the dimensionsof the channel, including the shape, width, length, and depth of thechannel, as described herein. In some embodiments, the depth of thechannel can be in the range of about 5 μm to about 3 mm, about 10 μm toabout 2 mm, or about 250 μm to about 1 mm. In some embodiments, thewidth of the channel can be in the range of about 100 μm to about 10 mm,about 250 μm to about 5 mm, about 500 μm to about 3 mm, or about 750 μmto about 1 mm. In certain preferred embodiments, the dimensions of thechannel are selected such that the target analyte is drawn into thechannel by capillary action.

The material of the metering stack 224 can, in some embodiments, formone or more fluid collecting chambers, or receiving chambers, along thelength of the channel. FIGS. 4A-5B illustrate embodiments of thechannel(s) 410 within the metering stack. As shown in FIGS. 4A-5B, theshape of the channel is not particularly limited and will vary based onthe requirements of the assay components in the corresponding assaystack. In some embodiments, the longitudinal cross section of thechannel, in a plane parallel to the layered components of the cartridge,can be a rectangle with a constant width or a combination of differentrectangular, circular, oval, and/or other shapes. FIG. 4A illustratesone embodiment in which the channel 410 has a rounded rectangularlongitudinal cross section. FIG. 4B illustrates a channel 410 with acombination of rectangular portions and circular portions, the latter ofwhich corresponds to receiving chambers located along the length of thechannel. The channel 410 can have an inlet 412 where the target analytesample can be inserted or drawn in (e.g., by capillary action) to fillthe channel 410.

The channel may also comprise one or more venting holes 411 that connectthe channel to the atmosphere. An example of such a configuration isillustrated in FIG. 4A, which shows channel 410 with a plurality ofventing holes 411 arranged along the length of channel 410 and a channelinlet 412. In addition, as indicated in FIG. 4B, channel 410 maycomprise one or more receiving chambers 413 for collecting andtemporarily storing one or more predetermined volumes of the targetanalyte prior to introducing the target analyte to the assay componentsin the assay stack. In certain embodiments, the predetermined volumes ofthe receiving chambers are such that they permit temporary storage andsubsequent delivery of a greater volume of the target analyte than achannel without such receiving chambers (cf. FIGS. 4A and 4B). Whenchannel 410 comprises one or more receiving chambers 413, such ventingholes 411 may be disposed along the portions of the channel 410 betweenthe receiving chambers 413, as illustrated in FIG. 4B. However, in otherembodiments, the venting holes may be coincident with the receivingchambers. In general, the venting holes release air bubbles that may betrapped in the channel during collection of the target analyte (e.g.,blood) thereby helping to distribute the target analyte sample in thechannel and facilitating subsequent distribution of the target analyte(or a portion thereof) to the assay components in the assay stack. Ifdesired, the metering stack can include one channel or multiple channelssharing a same channel inlet 412. FIG. 4C illustrates an embodiment withmultiple channels 410 in a metering stack, each equipped with ventingholes 411. The multiple channels 410 are fluidly connected and can befilled with the target analyte via common channel inlet 412.

FIGS. 5A-B illustrate two exemplary embodiments of channels according tothe invention. In FIG. 5A, channel 510 has venting holes 511 which arearranged along the length of channel 510 at uneven intervals 513, 514,and 515. When the target analyte is admitted into channel 510 viachannel inlet 512, this configuration permits different volumes of thetarget analyte to be delivered to the respective assay components. Ifdesired, the segment between venting holes 511 can hold a volume oftarget analyte in the range of 0.5-25 μl for each assay. Alternatively,as shown in FIG. 5B, channel 510 may be equipped with a plurality ofreceiving chambers 513, 514, and 515 which have different predeterminedvolumes to permit delivery of different volumes of the target analyte tothe respective assay components.

Preferably, the metering stack is designed to direct the target analytefluid to flow into the channel and into any receiving chamber(s) thatmay be present. In some embodiments, the channel can be formed of orcoated with a hydrophilic material, non-limiting examples of whichinclude 93210 hydrophilic PET (Adhesives Research, Glen Rock Pa.) or9984 Diagnostic Microfluidic Surfactant Free Fluid Transport Film, 9960Diagnostic Microfluidic Hydrophilic Film, or 9962 DiagnosticMicrofluidic Hydrophilic Film (3M Oakdale, Minn.). The channel can alsohave one or more porous or mesh material along at least some portions ofthe channel that allows at least a portion of the target analyte to bedispensed from the channel of the metering stack to contact assaycomponents in the assay stack. One non-limiting embodiment is shown inFIG. 6. FIG. 6 shows a plan view of metering stack layer 650 thatcomprises porous or mesh material 655 which is positioned such that itis aligned with the channel portion on its top surface and the assaydistribution ports and assay components on its bottom surface. In someembodiments, the porous or mesh material is selected such that the poresin such material separate the target analyte into a portion that is tobe delivered to the assay components and a portion that is not deliveredto the assay components. For example, when the target analyte is blood,the pores of the porous or mesh material may be of a size that issuitable for separating erythrocytes from other blood components, suchas plasma. In this way, when the cartridge is inserted into the assayreader to perform the assays, only plasma is delivered to the assaycomponents for analysis. Of course, combinations of porous or meshmaterials may be used such that the entire target analyte is deliveredto some of the assay components, while only portions of the targetanalyte may be delivered to other assay components. For example, thecombination of porous or mesh materials may allow only plasma to reachsome assay components, but allow for the delivery of all bloodcomponents to other assay components. In certain embodiments, thechannel can include a porous or mesh material at the bottom of thechannel. The porous or mesh material at the bottom of the channel can bea hydrophilic material or a material coated with a hydrophilic coating.In some embodiments, the porous or mesh material can have a pore sizebetween about 1 μm to about 500 μm. Advantageously, when the targetanalyte is blood, the pores of the porous or mesh material can be sizedto allow the porous or mesh material to hold the blood sample in thechannel without dripping during blood collection and to be absorbed bythe assay stack during the blood dispensing step which occurs uponinsertion of the cartridge into the assay reader.

In some embodiments, the porous or mesh material can also be used torelease air and prevent bubble formation during the time that channel isfilled with the target analyte. In some embodiments, the channel has around or octagonal transverse cross-section, as illustrated in FIG. 7A,and in such embodiments, the upper-most layer may have venting holesthat are off-set to the side. In FIG. 7A, the venting hole 711 can bedisposed at a 45-degree angle relative to the top surface of thecartridge and the mesh or porous material 703 can be positioned on thebottom of the channel. In such a configuration, air may escape fromventing hole 711 or through porous mesh 703 as the channel becomesfilled with the target analyte. In some embodiments, as illustrated inFIG. 7B, the venting holes 711 can be positioned on the bottom of themetering stack 704 within or near a mesh or porous material 703. Asillustrated in FIG. 7B, the channel of the metering stack may have oneside open to receive the target analyte. In some embodiments, thechannel inlet itself can be one of the venting holes of the channel and,optionally, the end of the channel opposite the channel inlet can beopen to the environment and serve as a venting hole for additional airto escape. In some embodiments, as illustrated in FIG. 7C, the ventingholes 711 can be positioned on the top the channel 710 and the mesh orporous material 703 can be positioned on the bottom of the channel 710.

FIG. 8 illustrates an exploded view of a metering stack according to oneexemplary embodiment of the invention. In FIG. 8, the metering stack 804is formed by assembling multiple layers. The first layer 841 can be aplastic sheet with a first side 842 in communication with thesurrounding environment when the cartridge is located outside the assayreader and a second side 843 that faces the assay stack. In someembodiments, the first layer 841 may be a cover layer or top layer ofthe metering stack. In preferred embodiments, first layer 841 may have ahydrophilic surface or coating on second side 843. Non-limiting examplesof suitable hydrophilic surfaces coatings includepolyvinylpyrrolidone-polyurethane interpolymer, poly(meth)acrylamide,maleic anhydride polymers, cellulosic polymers, polyethylene oxidepolymers, and water-soluble nylons or derivatives thereof, to name justa few. The presence of the hydrophilic surface or coating on second side843 helps to draw the target analyte into the channel, since most, ifnot all, of the target analytes are aqueous mixtures, such as blood. Thefirst layer 841 may include vent holes 811 positioned to align with thechannel 810 defined by the layers below. In FIG. 8A, for example, thevent holes 811 are aligned with the receiving chambers of channel 810 toallow air that otherwise would be trapped as an air bubble in thereceiving chamber during channel filling to escape efficiently into thesurrounding environment. It should be noted that the channel opening canalso serve as a vent hole, if desired. In certain preferred embodiments,the first layer 841 comprises polyethylene terephthalate (PET) with ahydrophilic coating on the second side 843 and with venting holes 811.

The second layer 844 is positioned below the first layer 841 on thesecond side or assay facing side of the first layer 841. The secondlayer 844 itself can be a combination of one or more layers asillustrated in FIG. 8. Regardless of whether second layer is comprisedof one layer or more than one layer, the second layer essentiallydefines the shape and size of channel in the metering stack, includingany receiving chambers that may be part of the channel. For example, thesecond layer 844 can be formed from one or more layers of polymericmaterial cut to define the volume and shape of the channel 810 that cancontain the target analyte. Other non-limiting methods of forming thechannel 810 include injection-molding, stamping, machining, casting,laminating, and 3-D printing. Combinations of such fabricationtechniques are also expressly contemplated by the invention. In theembodiment shown in FIG. 8, second layer 844 has a first side 847 facingthe first layer 841 and an opposite second side 848 that faces the assaystack. Furthermore, second layer 844 comprises adhesive layer 845 andplastic layer 846. Adhesive layer 845 fastens first layer 841 to plasticlayer 846. In some embodiments, the second layer 844 can be acombination of one or more layers of plastic sheet(s) 846 and adhesivelayers 845. Preferably, adhesive layer 845 or plastic layer 846 or bothare fabricated from materials which present a hydrophilic surface to theinterior surfaces of the channel 810 in order to facilitate thedistribution of the target analyte within channel 810. In someembodiments, the hydrophilic plastic sheet(s) can include a PET materialwith a channel 810 cut into it. If desired, channel 810 may include oneor more receiving chambers as shown in FIG. 8. Thus, the thickness andgeometry of channel 810 can control the volume of sample to becollected. The hydrophilic interior surfaces of the channel 810 allowthe metering stack to collect blood sample by capillary force. In someembodiments, the first layer 841 and the second layer 844 can be oneintegrated layer used in the metering stack.

In FIG. 8, third layer 849 can be formed from a hydrophobic adhesivelayer. Non-limiting examples of suitable materials for fabricating thirdlayer 849 include 3M 200MP adhesive or 3M 300MP adhesive (3M, Oakdale,Minn.). In preferred embodiments, the same channel geometry as channel810 is cut into the third layer to match channel 810 cut in the secondlayer. In some embodiments, the third layer 849 can have a first side851 facing the second layer 844 and an opposite side 852. In someembodiments, the third layer 849 can define the hydrophilic region in afourth layer 850 positioned below or on the second side 852 of the thirdlayer.

In some embodiments, the fourth layer 850 can be a hydrophilic mesh orporous material. In some embodiments, substantially all of the fourthlayer 850 can include the mesh or porous material as shown in FIG. 8. Inother embodiments, the hydrophilic mesh or porous material can be aportion of the fourth layer 850 as shown in FIG. 6. More specifically,FIG. 6 illustrates an embodiment of a fourth layer of the metering stackthat includes a mesh or porous material within the portion of the fourthlayer that aligns with the channel formed from the second and thirdlayers. In some embodiments, such as the example shown in FIG. 8, thefourth layer 850 can have a first side 853 facing the third layer 849and an opposite assay stack facing second side 854. The hydrophobicthird layer 849 can be positioned above the fourth layer 850. Thehydrophobic third layer 849 can be a hydrophobic adhesive layer todefine a wettable region of the mesh or porous material of the fourthlayer 850.

The method used to fabricate the metering stack is not particularlylimited, so long as it is compatible with the general manufacturingrequirements for medical devices. In certain embodiments, the layersthat constitute the metering stack are first fastened together as largemultilayer sheet or strip which is then subjected to stamping or cuttingprocesses to form the metering stack, including the channel and anyreceiving chambers that may be present. In some embodiments, the firstlayer 841 and second layer 844 can be combined in one piece of plasticmaterial with a hydrophilic surface forming the channel. In someembodiments, the third layer 849 and fourth layer 850 can be combined inone piece of patterned mesh made by printing or other method to definethe hydrophilic porous area. In some embodiments, the third layer is notused in the metering stack. Various other combinations of two or morelayers, as well as additional layers, are contemplated by variousembodiments.

In the assay systems of the invention, the assay reactions occur in theassay stack. In general, an assay stack comprises one or more “assaycomponents.” As used herein, the term “assay component” refers to one ormore of the active component and a passive supporting element or mask,including but not limited to the multiplexed assay pads. The numberassay pads in a particular assay component is not particularly limitedand is scalable to meet the assay requirements needed to diagnose thecondition of the patients for whom the assay stack is designed. Inpreferred embodiments, the top layers of the assay pads of a given assaycomponent align vertically with the appropriate regions of the channelin the metering stack above to ensure that a predetermined volume oftarget analyte, sufficient to perform the assay associated theparticular assay, is delivered to the assay pad. The assay pad can actas a sponge that draws the sample through the mesh of the metering stackinto the assay stack, for example through capillary action, gravity,etc. Therefore, once the metering stack and the assay stack are incontact with or within close proximity to each other, the fluid from thetarget analyte sample to be analyzed is directed to move into the assaypad, where it may encounter one or more chemical reagents required toperform the assay associated with the particular assay component. Ifdesired, the assay stack may comprise additional layers that contain thechemicals required for the completion of the assay. The number of layersrequired can depend on the number of chemical reactions that need totake place to complete the assay. For instance, some assays require asingle layer while others may require multiple layers. In variousembodiments, layers of the assay stack can be made of variously-shapedand variously-sized pads of different porous membrane materials,non-limiting examples of which include nylon, polyethersulphone(PES),nitrocellulose, cellulose filter paper, and glass fiber.

The type of assays that may be formed using the assay systems of theinvention are not particularly limited and can be any assay for whichthe required reagents can be stably incorporated into one or more assaypads and which can cause a change that can be detected by the assayreader. In preferred embodiments, the assay reactions cause a colorchange, which may be detected using the colorimetric detection methodsas described herein. In certain embodiments, the assays may be porousmaterial-based lateral flow assays, vertical flow assays, and/or acombination of lateral and vertical flow assays. In general, the targetanalyte is a biological fluid, non-limiting examples of which includeblood, saliva, sweat, urine, lymph, tears, synovial fluid, breast milk,and bile, or a component thereof, to name just a few. In certainpreferred embodiments, the target analyte is blood or a componentthereof (e.g., blood plasma). For example, in one embodiment, the assaysystems of the invention are useful for providing diabetic patients withpoint-of-care information regarding their blood composition, includingglucose level, hemoglobin A1C with eAG, C-peptide levels, creatininelevels, and the like. By way of example, glucose levels may be measuredby reaction with dinitrosalicylic acid, which results in a color changethat is proportional to the amount of glucose present. Alternatively,glucose levels in a target analyte may be analyzed by monitoring thedegree of change in yellow color characteristic of ferricyanide. Asanother example, the presence of creatinine can be detected by reactingcreatinine with a picrate, which results in a colored complex. In yetanother example, the assay systems may be used to evaluate the immunereactivity of blood platelets using a colorimetric assay chemistry basedon the reduction of tetrazolium salts. See, e.g., Vanhée D., et al, “Acolorimetric assay to evaluate the immune reactivity of blood plateletsbased on the reduction of a tetrazolium salt,” J. Immunological Methods,Vol. 159, Issues 1-2, February 1993 pp. 253-259. In other embodiments,when the target analyte is urine, the assay stack may comprise assaycomponents for measuring glucose, detecting uric acid, detectinghematuria, or detecting metabolites of illicit drugs, using assaychemistries as known in the art. For instance, when the target analyteis uric acid, an assay monitoring the reduction of cupric copper tocuprous copper, which may in turn complex with the neocuproine to form acolored material that is proportional in density to the concentration ofuric acid in the analyzed liquid. See, e.g., U.S. Pat. No. 3,992,158,which is incorporated by reference in its entirety.

In certain embodiments, one or more assay pads in an assay component donot contain any reagents for performing assays on the target analyte,but instead simply absorbs or adsorbs the target analyte to present itfor direct analysis using the assay reader of the invention. Forexample, in certain embodiments, the assay pad absorbs blood plasma thathas been separated from the red blood cells of the original bloodsample, using the methods described herein, and presents the bloodplasma for optical analysis by the assay reader to determine the extentto which hemolysis has occurred.

FIG. 9 illustrates an exemplary assay stack 906 according to oneembodiment of the invention. In FIG. 9, the assay stack 906 is formed ofmultiple layers, including one or more of the layers with activecomponents and a passive supporting element or mask. More specifically,in FIG. 9, assay stack 906 comprises assay stack cover layer 910 thatfeatures cut-out portion 911 that is aligned with the channel in theoverlying assay stack. Generally, assay stack cover layer 910 isfabricated from a polymeric material that provides rigidity to the assaystack and provides ease of handling during manufacturing of thecartridge. Furthermore, cut-out portion 911 allows the target analyte toflow past assay cover layer 910 towards the under assay components whenthe cartridge is inserted into the assay reader, as described herein. Insome embodiments, the assay stack 906 comprises a separation layer 961in the top most layer facing the metering stack, although this inventionalso expressly contemplates embodiments in which an assay stackcomprises a plasma separation layer which is not in the top most layerof the assay stack. It should be noted that the separation layer isoptional, and that in certain embodiments, the assay stack does notcomprise any separation layer. When present, separation layer 961 may beused to separate components of the target analyte to prevent undesirablecomponents of the target analyte from reaching the underlying assaycomponents. For example, when the target analyte is blood, theseparation layer 961 may be a plasma separation membrane that preventserythrocytes from reaching the assay components after the cartridge hasbeen inserted into the assay reader. This is advantageous because thestrong spectral absorption by the hemoglobin present in erythrocytes mayoverwhelm the color changes that occur at the assay pad after the assayis performed. Such a plasma separation membrane can be made of a varietyof materials, non-limiting examples of which include an asymmetricpolysulphone membrane, glass fiber, or cellulose. In some embodiments,the fabrication of the plasma separation membrane can include surfacetreatments for improved wettability and/or other properties. The plasmaseparation layer can be one continuous piece of membrane for all of theassay components, or multiple pieces of membrane material that may besame or different (or some combination thereof) for each of the assaypads in the assay component in the assay stack FIG. 9. In someembodiments, some of the assay pads of an assay component have acorresponding plasma separation layer, while other assay pads do nothave such a layer.

In FIG. 9, assay stack 906 includes assay component 920, which featuresmask support layer 930 with a plurality of cut-outs 931 that areconfigured to receive and immobilize assay pads 940 when the assay stack906 is assembled. Preferably, cut-outs 931 are positioned laterally inmask support layer 930 such that each of the assay pads 940 are alignedwith both the channel and the porous or mesh material of the meteringstack above in order to receive predetermined volumes of target analytesufficient to perform the assay reaction associated with the given assaypad. As shown in FIG. 9, in some embodiments, the assay stack 906 caninclude a second assay component 962 positioned below the plasmaseparation layer 961 and first assay component 920. The second assaycomponent 962 comprises a mask support layer 950 with a plurality ofcut-outs 951 that are configured to receive and immobilize assay pads963 when the assay stack 906 is assembled. Preferably, cut-outs 951 arepositioned to align assay pads 963 with assay pads 940, such that thetarget analyte will flow from assay pads 940 into assay pads 963. Assaypads 963 may comprise chemical reagents that are necessary to completethe assay reactions that are initiated when the target analyte flowsthrough the assay pads 940 of assay component 920. In other embodiments,one or more of assay pads 963 are non-functional pads that do not causeany further chemical reactions with the target analyte and merelytransmit the completed assay products to the bottom of the assay stackfor analysis by the assay reader. In some embodiments, assay pads 963serve as a detection indicator layer that provides informationcorresponding to the results of the assay performed. For example, assaypads 963 can include a visual indicator, such as a color change, toindicate the results of the assays. Furthermore, while assay stack 906in FIG. 9 contains only two assay components 920 and 962, it should beunderstood that the assay stack 906 may contain additional assaycomponents with assay pads that are impregnated with chemical reagentsthat are required to complete and/or report the results of a particularassay. For instance, the assay stack 906 can include any number of assaycomponents necessary to perform the analysis of the blood sample.Because some assays require more chemical steps than others, assaycomponents near the bottom of the assay stack may comprise morenon-functional assay pads which only serve to draw the completed assayproducts to the bottom of the assay stack, where the results may bedetected by the assay reader, as described herein.

Assay stack 906 in FIG. 9 also includes an assay bottom layer 970, whichis typically fabricated from a polymeric material to provide mechanicalstrength and ease of handling of assay stack 906 during themanufacturing process. In addition, assay bottom layer 970 typicallycomprises a plurality of detection ports 971 which are aligned with theassay pads of the assay stack and sized to permit interrogation of theassay results by the assay reader. For example, as described herein, theassay reader may probe the assay results by shining light of aparticular wavelength onto the assay pads of the bottommost assaycomponent in the assay and detecting the intensity or wavelength of thelight that is scattered from the assay pads.

FIG. 10 comprises a cartridge 1000 and an assay reader 1050 according toanother embodiment of the invention. Cartridge 1000 features a unitaryouter shell that includes handle portion 1005 and housing portion 1010.In this embodiment, handle portion 1005 is a long, thin tab that permitseasy handling of the cartridge 1000 by user, even when the user is usingonly one hand. As shown in FIG. 10, cartridge 1000 further comprisesmetering stack 1020 which is partially exposed when it is inserted intohousing portion 1010. In particular, channel inlet 1021 protrudesslightly from the housing portion 1010, such that the mouth of thechannel 1021 may be dipped into the target analyte, causing the targetanalyte to be drawn into the channel of metering stack 1020. A pluralityof venting holes 1023 are visible on the top layer of metering stack1020 and are aligned with the underlying channel of the metering stack1020. The venting holes 1023 prevent the formation of undesirable airbubbles in the channel while the channel is being filled with the targetanalyte. Not shown is a corresponding assay stack, which is positionedunderneath metering stack 1020.

After the channel of metering stack 1020 is filled with the targetanalyte, cartridge 1000 is inserted into assay reader 1050 (FIG. 11). Asshown in FIG. 10, cartridge 1000 features a slot 1015, which isconfigured to receive an internal tab that extends along thelongitudinal direction of assay reader 1050. The internal tab protrudesdownward into the receiving chamber 1052 assay reader 1050, creating arudimentary “lock and key” mechanism that makes it impossible for theuser to inadvertently insert cartridge 1000 upside down into assayreader 1050. In addition, when the cartridge 1000 is inserted into assayreader 1050 after the target analyte has been collected, the internaltab provides a compressive force that compresses together the meteringstack 1020 and the underlying assay stack, thereby initiating the assayreactions, as described herein.

FIG. 12 shows an exploded view of metering stack 1020 corresponding tothe embodiment of the cartridge shown in FIG. 10. Top layer 1201comprises a polymeric sheet with venting holes 1203 that are alignedwith the underlying channel in the metering stack. In certain preferredembodiments, the surfaces of top layer 1201 may be subjected to chemicaltreatments that facilitate the collection of the target analyte. Forinstance, when the target analyte is blood, it is advantageous to applya hydrophobic coating 1205 to the top surface of top layer 1201 and ahydrophilic coating 1206 to the bottom surface of top layer 1201. Inthis way, during blood collection, the hydrophobic top coating 1205 ontop layer 1201 prevents the blood from sticking to the exposed topsurface of the metering stack in the cartridge. On the other hand, thehydrophilic bottom coating 1206 of the top surface 1201 helps to drawthe blood into the metering stack when channel inlets 1221 and 1231 arebrought into contact with the blood, because the hydrophilic coatingallows the blood to wet the bottom surface of top layer 1201 as thechannel fills. The metering stack in FIG. 12 further includes an upperchannel layer 1220 and a lower channel layer 1230. Upper channel layer1220 includes channel 1222 which has channel inlet 1221 and threereceiving chambers 1223, 1224, and 1225. As shown in FIG. 12, receivingchamber 1225 is larger than receiving chambers 1223 and 1224 andtherefore capable of holding a larger volume of the target analyte whenthe chamber is filled. Lower channel layer 1230 comprises channel 1232with channel inlet 1231 and three receiving chambers 1233, 1234, and1235. In certain preferred embodiments, the top surface of lower channellayer 1230 is coated with a hydrophilic coating, which helps to draw thetarget analyte (e.g., blood) into channels 1222 and 1232 by permittingthe target analyte to wet the top surface of lower channel layer 1230.In certain preferred embodiments, the bottom (assay stack facing)surface of lower channel layer 1230 is coated with a hydrophobiccoating, which helps to localize the target analyte in receivingchambers 1233, 1234, and 1235 both during collection and when themetering stack is compressed by the assay reader to drive the targetanalyte into the underlying assay stack for analysis. At the bottom ofthe metering stack shown in FIG. 12 is porous hydrophilic layer 1250,which prevents the target analyte from contacting the underlying assaystack until the metering stack and assay stack are compressed togetheras a result of insertion into the assay reader.

In the “dual channel” configuration shown in FIG. 12, upper channellayer 1220 and lower channel layer 1230 are aligned such that they arein fluid communication when the metering stack is assembled. Preferably,the receiving chambers of upper channel layer 1220 and lower channellayer 1230 coincide, as shown in the FIG. 13A (top view) and FIG. 13B(perspective view). Thus, the channel and 1222 receiving chambers 1223,1224, and 1225 in upper channel layer 1220 serve as reservoirs ofadditional target analyte that can be delivered to correspondingreceiving chambers 1233, 1234, and 1235 in lower channel layer 1230. Inthis way, the additional target analyte in upper channel 1222 andcorresponding receiving chambers 1223, 1224, and 1225 ensure that asufficient amount of the target analyte is delivered to the underlyingassay stack when the cartridge is inserted into the assay reader. Thesize of the receiving chambers 1223, 1224, and 1225 may be varieddepending on the sample size requirements of the assay pads in the assaystack as described herein. For example, in the non-limiting embodimentshown in FIG. 12, receiving chamber 1225 in upper channel layer 1220 islarger than corresponding receiving chamber 1235 in lower channel layer1230. This permits different volumes of the target analyte to bedelivered as needed to the assay pads in the assay stack below, therebyproviding additional flexibility as to the types of assays that may beincorporated in the cartridges of the invention. In addition, the bottomof lower channel 1230 defines the fluid communication region between thechannel and the assay pad. In one embodiment, the fluid communicationregion is the same or smaller than the top surface area of the assaypad.

FIG. 14A shows metering stack 1400 and corresponding assay stack 1450before they are assembled together and inserted into the cartridge. Inthis non-limiting embodiment, metering stack 1400 has a dual-channelconstruction, with an upper channel layer and lower channel layer asshown in FIG. 12. When metering stack 1400 is assembled, upper channel1422 with receiving chambers 1423, 1424, and 1425 can be seen throughtop layer 1415. Venting holes 1440 are aligned with channel 1422 andpositioned between receiving chambers 1423 and 1424, between receivingchambers 1424 and 1425, and at the end of channel opposite the channelinlet 1421. Furthermore, receiving chambers 1423, 1424, and 1425 arealigned with the receiving chambers in the lower channel, but onlyreceiving chamber 1435 of the lower channel can be seen readily sincereceiving chambers 1423 and 1424 are the same size as and coincidentwith their corresponding receiving chambers in the lower channel. Thereceiving chambers in the lower channel layer are aligned withcorresponding assay pads 1452, 1454, and 1456 in assay stack 1450. Thisalignment is shown in FIG. 14B, which is a transverse cross-sectionalview of metering stack 1400 and assay stack 1450 through upper receivingchamber 1425 and lower receiving chamber 1435 after the metering stackand assay stack have been brought together.

FIGS. 15A-15D illustrate the operation of the cartridge in four stepsaccording to one exemplary implementation of the invention. For clarity,the embodiment shown in FIGS. 15A-15D comprises a metering stack withonly a single channel layer. It should be noted, however, that the basicprinciple of operation is similar for metering stacks with more than onechannel, as exemplified in FIGS. 12-14. FIG. 15A illustrates a schematiclongitudinal cross sectional view of metering stack 1504 during samplecollection. As shown in FIG. 15A, target analyte is distributed onsurface 1501, which may be the surface of a patient's skin when thetarget analyte is blood. During sample collection, the channel 1510 inthe metering stack 1504 can be filled with the target analyte (e.g.,blood) by exposing channel inlet 1505 to the target analyte 1502. Thetarget analyte 1502 is drawn into channel 1510 by capillary actionand/or by gravity, as indicated by arrow 1508. In some embodiments,pointing channel inlet 1505 of the channel 1510 of the metering stack1504 upwards can help blood flow into the channel 1510. As channel 1510begins to fill, the air in the channel 1510 is displaced by the targetanalyte 1502 and driven out of channel 1510 via vent holes 1511, asindicated for one vent hole by the black arrow labeled 1512. Inaddition, some of the air in channel 1510 may escape via the porous ormesh layer 1550 at the bottom of the metering stack. Furthermore, thepresence of the venting holes 1511 and/or porous or mesh layer 1550 alsopermits any air bubbles introduced into the channel by user error toescape before the sample collection is completed. Such air bubbles mayform, for example, if the user accidentally moves channel inlet 1505outside of the drop of target analyte 1502 on surface 1501 during samplecollection. In this way, the venting holes 1511 in metering stack 1504help to ensure that a predetermined volume of target analyte is reliablydelivered to the assay stack below. As shown in FIGS. 15A-D, themetering stack and assay stack can have extra space to allow overdraw ofthe sample without dispensing the extra sample to the assay pad. Inaddition, the channel 1510 in the metering stack 1504 may extend beyondthe last assay pad to act as a run-off area. If the user keeps fillingthe channel after the sample reaches the indicator location the excesssample can fill the extra volume in the channel beyond the last pad.

As described herein, the size of the pores or mesh in porous or meshlayer 1550 is selected to ensure that the target analyte does not leakthrough the porous or mesh layer 1550 during target analyte collection.In some embodiments, channel 1510 comprises a plurality of receivingchambers 1515 located along the length of the channel. Due to the natureof the longitudinal cross section shown in FIG. 15A, the positions ofthe receiving chambers 1515 are indistinguishable from the rest ofchannel 1510 and are therefore represented by the dotted lines, whichalso indicate that receiving chambers 1515 are positioned between ventholes 1511.

In the configuration shown in FIG. 15A, metering stack 1504 is separatedfrom assay stack 1506 by spacing 1525, which may achieved by inserting acompressible spacing material (not shown for clarity) between themetering stack and the assay stack. The compressible spacing material ispositioned in spacing 1525 such that it does not interfere with thetransfer of the target analyte 1502 from the metering stack to theunderlying assay stack 1506. Assay stack 1506 includes a plurality ofassay pads 1530 which contain reagents that interact with the targetanalyte 1502 to provide an assay result when the target analyte istransferred from metering stack 1504 to assay stack 1506. In thisexemplary embodiment, assay stack 1506 also features separation layer1531 which may be used to prevent a portion of the target analyte fromreaching the assay pads 1530 (e.g., red blood cells, if the targetanalyte is blood). Separation layer 1531 may also contain assay reagentsthat interact with the target analyte 1502. In addition, for each assaypad 1530, separation layer 1531 may be comprised of different materialswith different thicknesses and/or reagents contained within, although inthis illustrative embodiment, separation layer 1531 is one continuouspiece. The assay stack includes one or a plurality of assay pads, whichmay be used for different functions, non-limiting examples of whichinclude separation of analyte components, assay reactions, or acombination thereof. In some embodiments, the assay stack contains onlyone or more assay pad with assay reagents, and the assay pads do notfunction as a separation layer. In certain embodiments, one or moreassay pads function as separation layers and do not contain any assayreagents. Of course, the invention also contemplates embodiments wherethe assay stack contains assay pads that contain reagents and alsofunction as a separation layer. For the purposes of illustration, a gap1532 is shown between the separation layer 1531 and the assay pads 1530.However, in many (if not most) embodiments, assay pads 1530 will be indirect contact with separation layer 1531, so that the target analytewill be wicked directly into the assay pads when the metering stack andthe assay stack are brought together.

The filling of the channel 1510 with target analyte can be judged byindicators on the metering stack 1504. In some embodiments, theindicator can be a visual indicator. For example, the presence of thetarget analyte (e.g., blood) inside the channel may be visible through atransparent material above or surrounding the channel or at the end ofthe channel. In other embodiments, the visible indicator can be anindicator initiated when target analyte reaches a particular location inthe channel. For example, the visible indicator can be initiated whentarget analyte reaches the end of the channel. For example, when thetarget analyte is blood, the top layer of the metering stack 1504 can bedesigned so that the blood is visible only through a slit at a givenlocation of the channel 1510. Once the blood reaches this location theuser can see a “red slit,” a “red line,” or any other indicator whichcan be used as a visual cue for a user to know when to stop collectingthe sample. In some embodiments, the indicator can be a light emittingdiode (LED) that activates when the blood reaches a set point. In someembodiments, the indicator can be activated by electrodes. Theelectrodes can trigger or activate an alarm, light, or other indictorwhen the electrodes are in contact with blood in the channel.

FIG. 15B shows metering stack 1504 and assay stack 1506 after thechannel 1510 has been filled with target analyte 1502 and inserted intothe assay reader, causing the compression of the spacer material betweenthe metering stack 1504 and the assay stack 1506 the removal of gap1525. In this way, metering stack 1504 and the assay stack 1506 arebrought together. At this point, target analyte 1502 is permeatingthrough porous or mesh layer 1550 and separation layer 1531, but has notreached assay pads 1530, as shown by the absence of any target analytein gap 1532. In certain embodiments, due to the design of metering stack1504, target analyte 1502 reaches all of the assay pads at essentiallythe same time. This is illustrated schematically in FIG. 15C, whichshows target analyte portions 1502 a, 1502 b, 1502 c, and 1502 dpermeating porous or mesh layer 1550 and separation layer 1531 andcontacting assay pads 1530 in parallel. The synchronization of theassays in the assay stack is advantageous, because many assays requirethe reagents to react for a certain time before valid assay results canbe obtained. By providing a well-defined starting point for the assays,the invention provides a reliable, repeatable system assay system forperforming different assays at the same time. It should be noted,however, that the invention also specifically contemplates embodimentswhere the start of the assays is not synchronized. For example, incertain embodiments, the assay reader comprises sensors which arecapable of detecting the actual starting time and ending time bymonitoring a signal change as described herein, a non-limiting exampleof which includes a change in color. When the target analyte 1502contacts assay pads 1530, the target analyte is drawn or wicked into theassay pads by capillary action and/or gravity, as indicated by the blackarrows in FIG. 15C. At this point, vent holes 1511 allow ambient air toenter channel 1510, thereby preventing the formation of a partial vacuumthat would otherwise be caused by the absorption of the target analyte1502 from the channel 1510 into the assay pads 1530 below.

In FIG. 15D, a substantial portion of target analyte 1502 has been drawnout of channel 1510 toward the underlying assay pads 1530 below,resulting in the emptying of a substantial portion of channel 1510. Incertain embodiments, portions of the target analyte in the channel abovethe assay pads “break off” from the target analyte in the rest of thechannel, due to the wicking action of the assay pads below. This mayresult in some target analyte 1502 being left behind in the channel 1510in regions which are not directly over an assay pad, such as near theends of the channel, between the end of the channel and the nearestventing hole (see FIG. 15D). Assay pads 1530 receive the portions of thetarget analyte 1502 that can pass through the separation layer 1531.This portion of the target analyte then undergoes assay reactions inassay pads 1530. In certain embodiments, the assay pads undergo a change(e.g., a color change) which can be detected by the assay reader, asdescribed herein.

FIG. 16A shows a schematic drawing of an assay reader, in longitudinalcross-section, according to one non-limiting embodiment of theinvention. In FIG. 16A, assay reader 1600 includes cartridge receivingchamber 1610 which houses the cartridge when it is inserted as indicatedby arrow 1605. Tab 1615 runs longitudinally along assay reader 1600 andextends into cartridge receiving chamber 1610. Tab 1615 is configured toinsert into a slot at the top of the cartridge, such as slot 228 in FIG.2C or slot 1015 in FIG. 10, when the cartridge is inserted into theassay reader. In addition, the spacing 1625 between the bottom edge oftab 1615 and support surface 1620 is set such that when the cartridge isinserted, tab 1615 compresses the metering stack and the assay stacktogether, thereby causing the target analyte to flow from the meteringstack into the assay stack and initiating the assay reactions. Incertain embodiments, the assay reader may comprise a snap-fit mechanismthat locks the cartridge in place once it has been fully inserted intothe assay reader. This is advantageous because it prevents the user fromaccidentally removing the cartridge from the assay reader before theassays are complete, which could adversely affect the accuracy of theassay results. In some embodiments, assay reader 1600 also comprisessensors 1642 a and 1642 b, which detect and time the insertion of thecartridge. For example, as the cartridge is inserted into cartridgereceiving chamber 1610 and begins to engage with tab 1615, the bottomsurface of the cartridge may pass over sensor 1642 a, which is detectedby appropriate electronics as the beginning of the insertion of thecartridge. The second sensor, 1642 b, is located further inside theassay reader 1600 and detects the presence of the cartridge when thecartridge is fully inserted as well as the time at which full insertionoccurred. Assay reader 1600 may then compare the overall time forinsertion of the cartridge to determine if the insertion of thecartridge was timely and proper. In this way, assay reader will notperform any assay readings in situations where (1) the cartridge wasonly partially inserted, or (2) the cartridge was partially inserted,removed, and inserted again. Either case could give inaccurate assayreadings, due to incomplete compression of the metering stack and assaystack, resulting in incomplete delivery of the required amount of targetanalyte to the assay pads in the assay stack.

In the exemplary embodiment shown in FIG. 16A, assay reader 1600 detectsthe results of the assay by detecting the color change of the assay padcaused by the assay reactions. To achieve this, assay reader 1600comprises a plurality of light sources (not shown in thiscross-sectional drawing) and light detection elements 1650 arrayedwithin assay reader 1600 such that they align with the assay pads of thecartridge when the cartridge is fully inserted. In order for lightdetection elements 1650 to be able to detect the color of the assaypads, support surface 1650 may be equipped with one or more apertures orbe fabricated from a transparent material that allows light to penetratetherethrough. FIG. 16B shows a schematic illustration of a longitudinalcross-section of assay reader 1600 with cartridge 1602 fully inserted.Cartridge 1602 includes metering stack 1604 and assay stack 1606, whichare compressed together by tab 1615 such that the target analyte isdelivered from the metering stack 1604 to the assay pads 1630. Assaypads 1630 are aligned with light detection elements 1650. Note, however,that assay reader 1600 may comprise an additional light detectionelement 1650 a without a corresponding assay pad 1630. The presence ofadditional light detection elements, such as light detection element1650 a, allow the assay reader to be used with different types ofcartridges for different assays, particularly cartridges that may bedesigned to perform more assays, as well as to identify the differenttypes of cartridges for the different assays.

FIG. 17A shows a schematic drawing of a transverse cross-section of theassay reader shown in FIG. 16. In FIG. 17A, assay reader 1700 includes atab 1715 that extends into cartridge receiving chamber 1710 to engagewith a slot on the cartridge to compress the metering stack and theassay stack against support surface 1720 to initiate the assayreactions. Light sources 1760 a and 1760 b provide light for detectingthe assay results and are positioned near light detection device 1750.As illustrated in FIG. 17A, light sources 1760 a and 1760 b providelight to analyze the assay pad corresponding to light detection element1750. In general, it is advantageous to dedicate one or more lightsources to each light detection element in order to ensure that thephoton flux onto the light detection element is sufficient to obtain anaccurate reading. In some embodiments, the light sources dedicated to aparticular light detection element have the same output spectrum. Inother embodiments, however, the light sources corresponding to a givenlight detection element produce different output spectra. For instance,the light sources may be light emitting diodes (LEDs) that producedifferent colors of light. For example, when the target analyte isblood, it may be useful to use light sources that can generatebichromatic pairs (600 nm/570 nm) to detect the presence of undesirablehemolysis. In general, it is advantageous to include optical elements todirect the light and/or reduce the amount of light scattering in theassay reader. In some embodiments, the optical elements are aperturesthat only allow light emanating from the light source that isline-of-sight to the respective assay pad to reach the assay pad. Forexample, in FIG. 17A, light source 1760 a is limited by aperturedefining members 1770 a and 1771 a such that only the light from lightsource 1760 a that passes through aperture 1773 a will reach the assaypad and subsequently be detected by light detection device 1750.Similarly, light source 1760 b is limited by aperture defining members1770 b and 1771 b, such that only the light from light source 1760 bthat passes through aperture 1773 b will reach the assay pad andsubsequently be detected by light detection device 1750. In preferredembodiments, aperture defining members 1770 a, 1770 b, 1771 a, and 1771b are fabricated from a black matte material to reduce the amount ofundesirable scattering when light sources 1760 a and 1760 b are turnedon. Furthermore, in this embodiment, light detection device 1750 locatedin a housing that is comprised of aperture defining members 1771 a and1771 b that only permit light that passes through aperture 1772 to reachlight detection device 1750. If desired, the aperture 1772 may be fittedwith a filter to admit only light of a predetermined wavelength orwavelength range for detection by light detection device 1750. This maybe useful, for example, when the light sources are equipped to provideonly white light for colorimetric analysis. In addition the light fromlight sources 1760 a and 1760 b and the light to be detected by lightdetection device 1750 may be directed or manipulated using opticalelements such as lenses, filters, shutters, fiber optics, light guides,and the like without departing from the spirit and the scope of theinvention.

FIG. 17B shows a schematic illustration of the operation of the assayreader described in FIG. 17A. In FIG. 17B, a cartridge comprisingmetering stack 1704 and assay stack 1706 are inserted into cartridgereceiving chamber 1710 of assay reader 1700. Tab 1715 compressesmetering stack 1704 and assay stack 1706 against support surface 1720 tocause the target analyte to flow from the channel 1712 into assay pad1730. As noted previously, assay reader 1700 may be fitted with sensorsto confirm that the cartridge has been inserted correctly and in atimely manner. Assay reader 1700 may also be pre-programmed beforesample collection, either by the user or during the manufacturingprocess, to illuminate the assay pads at the appropriate time based onthe type of cartridge being used. In this way, assay reader 1700collects assay data from assay pad 1730 only when the assay iscompleted. Alternatively, if desired, assay reader 1700 may beconfigured to collect assay data from assay pad 1730 during the entireassay reaction after the cartridge has been inserted. As shown in FIG.17B, light source 1760 a provides light beam 1780 a, which impinges onthe bottom face of assay pad 1730. Similarly, light source 1760 bproduces light beam 1780 b, which may impinge on the bottom of the assaypad 1730 at the same time as light beam 1760 a or a different time,depending on the requirements of the assays being detected.

FIG. 18 shows a block diagram of a sensor configuration inside an assayreader according to one exemplary embodiment of the invention. In FIG.18, four assay pads (identified by reference numerals 1841, 1842, 1843,and 1844) have completed their assay reactions with the target analyte,undergone the respective color changes, and are ready for colorimetricanalysis. Note that, if desired, this configuration can also be used tocollect data from the four assay pads to monitor the progress of theassay reactions. Input signal 1801 from a first microcontrollerserial-peripheral interface bus (MCU SPI Bus) enters digital-to-analogconverter unit 1810, which comprises individual digital-to-analogconverters 1811, 1812, 1813, and 1814 that independently control currentsources 1821, 1822, 1823, and 1824. These current sources, in turn,power light sources 1831, 1832, 1833, and 1834 respectively. In someembodiments, input signal 1801 may be sent by a timing circuit at apredetermined time after the insertion of the cartridge into the assayreader. In such embodiments, the predetermined time corresponds to theknown time or times for the assay reactions in the assay pads to reachcompletion. In some preferred embodiments, the light sources 1831, 1832,1833, and 1834 are activated at the same time to measure theassay-induced color change of assay pads 1841, 1842, 1843, and 1844simultaneously in a multiplexed mode. However, this invention alsocontemplates operating all of the light sources separately andsequentially, or some simultaneously and some separately, depending onthe timing requirements of the assays in the cartridge.

In this non-limiting example, each of light sources 1831, 1832, 1833,and 1834 comprises individual three light emitting diodes (LEDs) whichmay be the same or different colors, depending on the requirements ofthe assay and any optical elements that may be present in the assayreader. For example, in certain embodiments, the three LEDs in aparticular light source (e.g., 1831) may be red, green and blue (RGBLEDs), such that the light impinging on the assay pad is white lightwhen all three LEDS are activated. Of course, the light sources are notlimited to any particular number or type of LEDs or other lightgenerating devices. More generally, the light sources that are useful inthe assay readers of the invention are not particularly limited, so longas they provide light of suitable wavelength(s) and brightness for thelight detection element to make an accurate reading of the colored lightreflected from the assay pad. In certain non-limiting embodiments, thelight sources are light emitting diodes (LEDs), organic light emittingdiodes (OLEDs), active matrix organic light emitting diodes (AMOLEDs) orlasers. For example, the light source may be only one LED that hassufficient brightness and the proper wavelength to allow colorimetricanalysis of an assay reaction in a given assay pad. In certainembodiments, the light sources may produce light of specificwavelengths. As one non-limiting example, when the target analyte isblood (with erythrocytes removed), a bichromatic light source thatproduces light at 570 nm and 600 nm may be used to detect the presenceof heme on a non-functional (i.e., assay reagent-free) assay pad, whichis indicative of undesirable hemolysis in the patient. Alternatively,the light source may be a broadband source that is paired with one ormore narrow bandpass filters to select light of certain desiredwavelength(s). Typically, the light sources produce light in the visibleregion of the electromagnetic spectrum (i.e., wavelength between 400-700nm) although this invention also contemplates light sources that produceelectromagnetic radiation in the infrared (700 nm to 10⁶ nm) orultraviolet regions (10 nm-400 nm) of the electromagnetic spectrum, solong as they are paired with the appropriate light detection devices.Combinations of different light sources are also expressly contemplatedby the invention.

In FIG. 18, element 1840 is a schematic representation of opticalelements that optionally may be present in the optical path between thelight sources 1831, 1832, 1833, and 1834 and assay pads 1841, 1842,1843, and 1844. When desired, one or more optical elements may belocated between the light source and its corresponding assay pad todirect the light, focus the light, reduce undesirable scattering, selectone or more wavelengths for assay detection, or some combinationthereof. Non-limiting examples of such optical elements includeapertures, lenses, light guides, bandpass filters, optical fibers,shutters, and the like. Similarly, element 1842 represents opticalelements that optionally may be present in the optical path betweenassay pads 1841, 1842, 1843, and 1844 and corresponding light detectiondevices 1851, 1852, 1853, and 1854. These optical elements may be usedto manipulate the light upstream of the light detector devices in amanner similar to that described for element 1840. It is to beunderstood that different types and numbers of optical elements may beused for each combination of light source, assay pad, and lightdetection device. Light detecting devices 1851, 1852, 1853, and 1854detect the light from the assay pads 1841, 1842, 1843, and 1844. In thisnon-limiting example, the light detecting devices are photodiodes. Moregenerally, the type of light detection device is not particularlylimited, provided that it is capable of detecting the light that isreflected from the assay pads used for colorimetric measurement of theassay results. Other examples of suitable light detection elementsinclude photodiode arrays, CCD chips, and CMOS chips. The outputs fromphotodiodes 1851, 1852, 1853, and 1854 are sent to transimpedanceamplifier/low pass filter elements 1861, 1862, 1863, and 1864, whichconvert the current signal from the photodiodes to a voltage output,while filtering unwanted signal components. The output from elements1861, 1862, 1863, and 1864 are sent to analog-to-digital converter unit1870, which comprises multiplexer unit 1871, gain 1872, andanalog-to-digital converter 1873. The output of analog-to-digitalconverter unit 1870 may be sent to a component 1880, which may be asecond MCU SPI bus, a transmitter, or a processor. In certainembodiments, the transmitter allows for hardwired or wirelessconnectivity (e.g., Bluetooth or Wi-Fi) with a personal computer, mobiledevice, or computer network. In one particularly useful embodiment, theassay results are transmitted to the user's mobile device or personalcomputer, where they are displayed in a graphical user interface (GUI).If desired, the GUI may display prior assay results, in addition to thecurrent results, in order to provide the user with information regardingthe overall trends in the results of the assays. For example, if theuser is diabetic, the GUI may plot the glucose levels measured by theassay reader as a function of time to allow the user to determinewhether blood glucose level is being properly controlled. In addition,the assay results may be transmitted from the user's mobile device orcomputer to a computer network, such as one belonging to the user'sphysician. In this way, the assay systems of the invention can allow auser's physician to monitor a patient closely, by providing up-to-datemedical information from the assay results obtained by the assay reader.

It should be noted that the optical detection systems described in theforegoing correspond to some exemplary embodiments of the system, butthat the invention expressly contemplates other types of detectionsystems as well. In general, any detection system which corresponds to asignal change caused by an assay reaction may be used in connection withthe assay reader of the invention. Thus, for example, in certainembodiments, the detection system is an optical detection system that isbased on chemiluminescence. In such embodiments, light sources such asLEDS and OLEDS are not required to detect a color change caused by theassay reaction in the assay pads. Rather, the signal change may becaused by the reaction of an oxidative enzyme, such as luciferase, witha substrate which results in light being generated by a bioluminescentreaction. In another exemplary embodiment, the signal change caused bythe assay reaction may be detected by electrochemical reaction. As onenon-limiting example, the presence of glucose in a biological sample maybe tested using an electrochemical enzymatic sensor, which consists of aplatinum electrode coated with a glucose oxidase layer that is separatedfrom the biological sample by a semipermeable membrane. Such sensorshave been reported, for example, by Mor et al., in an article entitled“Assay of glucose using an electrochemical enzymatic sensor” AnalyticBiochemistry, Vol. 79, Issues 1-2, May 1977 pp. 319-328, which is herebyincorporated by reference in its entirety.

FIG. 19 shows a flowchart that illustrates a method of using of theassay system according to one embodiment of the invention to perform aplurality of assays. The method includes step 1910, which involvesreceiving a target analyte into a first channel in a cartridge. Step1920 involves inserting the cartridge into an assay reader, therebycompressing the cartridge to expose at least one component of the targetanalyte stored in a first channel to a plurality of assay pads in thecartridge simultaneously to cause a plurality of assay reactions. Step1930 involves detecting one or more signal changes associated with theplurality of assay reactions.

FIG. 20 shows a flowchart that illustrates a method of fabricating acartridge according to one embodiment of the invention. The methodincludes the steps of obtaining a first layer comprising (1) a layer ofpolymeric material with a channel formed therein and (2) a porous ormesh material attached on a bottom surface of the polymeric materialsuch that the channel is bounded on a bottom surface by the porous ormesh material; (step 2010) and obtaining a second layer comprising twoor more assay pads, each comprising a reagent for performing an assay onthe target analyte or a portion thereof (step 2020). The method alsoincludes the step of obtaining a compressible intermediate layercomprising a compressible material (step 2030). It is to be understoodthat steps 2010, 2020, and 2030 can occur independently, and notnecessarily stepwise or sequentially. In addition, in certainembodiments, the compressible intermediate layer can be attached to, orbe a part of the first or second layer. The method also includes thestep of combining the first layer, compressible intermediate layer, andsecond layer in a cartridge housing such that the compressibleintermediate layer separates the first layer and the second layer whenthe compressible intermediate layer is in an uncompressed state, and thechannel is aligned with the two or more assay pads in a directionperpendicular to the first layer (step 2040).

While various embodiments of the invention have been described above, itshould be understood that they have been presented by way of exampleonly, and not by way of limitation. Likewise, the various diagrams maydepict an example architectural or other configuration for thedisclosure, which is done to aid in understanding the features andfunctionality that can be included in the disclosure. The disclosure isnot restricted to the illustrated example architectures orconfigurations, but can be implemented using a variety of alternativearchitectures and configurations. Additionally, although the disclosureis described above in terms of various exemplary embodiments andimplementations, it should be understood that the various features andfunctionality described in one or more of the individual embodiments arenot limited in their applicability to the particular embodiment withwhich they are described. They instead can be applied, alone or in somecombination, to one or more of the other embodiments of the disclosure,whether or not such embodiments are described, and whether or not suchfeatures are presented as being a part of a described embodiment. Thusthe breadth and scope of the present disclosure should not be limited byany of the above-described exemplary embodiments.

Unless otherwise defined, all terms (including technical and scientificterms) are to be given their ordinary and customary meaning to a personof ordinary skill in the art, and are not to be limited to a special orcustomized meaning unless expressly so defined herein. It should benoted that the use of particular terminology when describing certainfeatures or aspects of the disclosure should not be taken to imply thatthe terminology is being re-defined herein to be restricted to includeany specific characteristics of the features or aspects of thedisclosure with which that terminology is associated. Terms and phrasesused in this application, and variations thereof, especially in theappended claims, unless otherwise expressly stated, should be construedas open ended as opposed to limiting. As examples of the foregoing, theterm ‘including’ should be read to mean ‘including, without limitation,’‘including but not limited to,’ or the like; the term ‘comprising’ asused herein is synonymous with ‘including,’ ‘containing,’ or‘characterized by,’ and is inclusive or open-ended and does not excludeadditional, unrecited elements or method steps; the term ‘having’ shouldbe interpreted as ‘having at least;’ the term ‘includes’ should beinterpreted as ‘includes but is not limited to;’ the term ‘example’ isused to provide exemplary instances of the item in discussion, not anexhaustive or limiting list thereof; adjectives such as ‘known’,‘normal’, ‘standard’, and terms of similar meaning should not beconstrued as limiting the item described to a given time period or to anitem available as of a given time, but instead should be read toencompass known, normal, or standard technologies that may be availableor known now or at any time in the future; and use of terms like‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words ofsimilar meaning should not be understood as implying that certainfeatures are critical, essential, or even important to the structure orfunction of the invention, but instead as merely intended to highlightalternative or additional features that may or may not be utilized in aparticular embodiment of the invention. Likewise, a group of itemslinked with the conjunction ‘and’ should not be read as requiring thateach and every one of those items be present in the grouping, but rathershould be read as ‘and/or’ unless expressly stated otherwise. Similarly,a group of items linked with the conjunction ‘or’ should not be read asrequiring mutual exclusivity among that group, but rather should be readas ‘and/or’ unless expressly stated otherwise.

Where a range of values is provided, it is understood that the upper andlower limit, and each intervening value between the upper and lowerlimit of the range is encompassed within the embodiments.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. The indefinite article “a” or “an” does not exclude aplurality. A single processor or other unit may fulfill the functions ofseveral items recited in the claims. The mere fact that certain measuresare recited in mutually different dependent claims does not indicatethat a combination of these measures cannot be used to advantage. Anyreference signs in the claims should not be construed as limiting thescope.

It will be further understood by those within the art that if a specificnumber of an introduced claim recitation is intended, such an intentwill be explicitly recited in the claim, and in the absence of suchrecitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should typically be interpreted to mean “atleast one” or “one or more”); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of “two recitations,” without othermodifiers, typically means at least two recitations, or two or morerecitations). Furthermore, in those instances where a conventionanalogous to “at least one of A, B, and C, etc.” is used, in generalsuch a construction is intended in the sense one having skill in the artwould understand the convention (e.g., “a system having at least one ofA, B, and C” would include but not be limited to systems that have Aalone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). In those instances where aconvention analogous to “at least one of A, B, or C, etc.” is used, ingeneral such a construction is intended in the sense one having skill inthe art would understand the convention (e.g., “a system having at leastone of A, B, or C” would include but not be limited to systems that haveA alone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). It will be furtherunderstood by those within the art that virtually any disjunctive wordand/or phrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or “B” or “A and B.”

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification are to be understood as beingmodified in all instances by the term ‘about.’ Accordingly, unlessindicated to the contrary, the numerical parameters set forth herein areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims in any application claiming priority to the present application,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

All of the features disclosed in this specification (including anyaccompanying exhibits, claims, abstract and drawings), and/or all of thesteps of any method or process so disclosed, may be combined in anycombination, except combinations where at least some of such featuresand/or steps are mutually exclusive. The disclosure is not restricted tothe details of any foregoing embodiments. The disclosure extends to anynovel one, or any novel combination, of the features disclosed in thisspecification (including any accompanying claims, abstract anddrawings), or to any novel one, or any novel combination, of the stepsof any method or process so disclosed.

Various modifications to the implementations described in thisdisclosure may be readily apparent to those skilled in the art, and thegeneric principles defined herein may be applied to otherimplementations without departing from the spirit or scope of thisdisclosure. Thus, the disclosure is not intended to be limited to theimplementations shown herein, but is to be accorded the widest scopeconsistent with the principles and features disclosed herein. Certainembodiments of the disclosure are encompassed in the claim set listedbelow or presented in the future.

What is claimed is:
 1. An analyte collection device, comprising: ametering stack comprising a first channel, wherein the first channelcomprises a porous or mesh material and one or more venting holes; anassay stack comprising one or more assay elements, at least one of theassay pads comprising a reagent for performing an assay; and a spacingelement positioning the metering stack and the assay stack apart fromeach other when in an uncompressed state and allowing the metering stackand the assay stack to come into contact when in a compressed state,wherein the porous or mesh material permits flow from the metering stackto the assay stack only when in the compressed state.
 2. The device ofclaim 1, wherein the first channel further comprises one or morechambers, at least one of the chambers configured to deliver apredetermined volume of analyte to the assay stack when coming incontact with the assay stack.
 3. The device of claim 1, wherein thefirst channel further comprises a plurality of venting holes along thefirst channel.
 4. The device of claim 1, wherein the assay is performedon a biological fluid selected from the group consisting of blood,saliva, sweat, urine, lymph, tears, synovial fluid, breast milk, serum,plasma, bile, or a component thereof.
 5. The device of claim 4, whereinthe assay is performed on blood and the assay stack comprises aseparation layer that prevents erythrocytes from contacting the one ormore assay pads.
 6. The device of claim 1, wherein the first channelcomprises a hydrophilic coating on at least part of an interior surface.7. The device of claim 1, wherein the assay comprises a color change toat least one of the one or more assay elements.
 8. The device of claim1, wherein the assay comprises at least one of a chemiluminescentreaction, an electrochemical reaction, or a colorimetric reaction. 9.The device of claim 1, wherein the first channel is in fluidcommunication with a reservoir that is adapted to receive an analyte.10. The device of claim 1, wherein the cartridge further comprises asecond channel in fluid communication with the first channel along atleast a portion of the length of the first channel.
 11. The device ofclaim 10, wherein the second channel further comprises at least onechamber in fluid communication with a chamber of the first channel. 12.The device of claim 11, wherein the at least one chamber of the secondchannel has a larger volume than a volume of the corresponding chamberof the first channel.
 13. A method of fabricating a cartridge, themethod comprising: obtaining a first layer comprising (1) a firstmaterial with a channel formed therein, wherein the channel comprises atleast one venting hole, and (2) a porous or mesh material attached on abottom surface of the first material; obtaining a second layercomprising one or more assay elements, at least one of the assayelements comprising a porous material capable of absorbing analyte, anda reagent for performing an assay; obtaining a spacing mechanism; andcombining the first layer, the spacing mechanism, and second layer in ahousing such that the spacing mechanism separates the first layer andthe second layer when in an uncompressed state, and the channel isaligned with the two or more assay elements in a direction perpendicularto the first layer.
 14. The method of claim 13, further comprisingcoating an interior surface of the channel with a hydrophilic coating.15. The method of claim 13, further comprising stacking a cover layercomprising a plurality of vent holes over the first layer, such that thevent holes are aligned with the channel in a direction perpendicular tothe first layer.
 16. The method claim 13, wherein the spacing mechanismprovides separation of the first layer and the second layer when in anuncompressed state.
 17. A cartridge for collecting blood for testing,the cartridge comprising: a metering stack configured to receive anddistribute the blood along a first channel, wherein the first channelhas a portion that comprises a porous or mesh material and one or moreventing holes; an assay stack comprising one or more assay pads, atleast one of the one or more assay pads comprising a reagent forperforming an assay on the blood or a portion thereof; and a separationmechanism preventing fluid communication between the metering stack andthe assay stack when in an uncompressed state and allowing the meteringstack and the assay stack to come into fluid communication when in acompressed state, wherein the porous or mesh material permits the bloodor a portion thereof to flow from the metering stack to the assay stackonly when in the compressed state.
 18. The cartridge according to claim17, wherein the at least one assay pad comprises a reagent for measuringthe concentration of at least one of glucose, Hemoglobin, Cholesterol,Triglycerides, and creatinine in the blood.
 19. The cartridge accordingto claim 17, wherein the metering stack and the assay stack areseparated in the uncompressed state and brought into contact in thecompressed state.
 20. The cartridge according to claim 17, wherein theseparation mechanism includes a spacing element positioned between themetering stack and the assay stack, the spacing element beingcompressible to permit fluid communication between the metering stackand the assay stack.